Project Summary/Abstract In addition to supplying nutrients to support growth, breast milk also has important immune functions that provide protection against infection and influence the maturation of the neonatal immune system. Many epidemiological studies have demonstrated that breastfeeding protects against a variety of allergic and autoimmune diseases and several rodent models have demonstrated that breast milk fosters lasting immune tolerance to specific environmental antigens. In this application, we focus on the potential immune modulating effects of butyrophilin (BTN1A1). BTN1A1 is structurally related to the B7 co-signaling molecules that regulate T cell activation or tolerance. It is expressed in the apical membrane of mammary epithelial cells during lactation and participates in the trafficking and secretion of intracellular lipid droplets from mammary epithelial cells into milk. We now present preliminary data showing that the BTN1A1 found in milk also regulates T cell responsiveness, both within the neonatal gut as well as systemically. Furthermore, the effects of BTN1A1 on T cell responsiveness persist well after the exposure to BTN1A1 ends at weaning. Therefore, our hypothesis is that that BTN1A1 contributes to the development of immune tolerance in neonates and is required for breast milk to inhibit the development of reactive airway disease to the environmental antigen, ovalbumin. The goal of this R21 application is to test this hypothesis through two “high-risk” but “high-reward” aims. Aim 1 will begin to characterize the molecular pathways by which milk BTN1A1 inhibits T cell activation by utilizing the power of unbiased, high-throughput sequencing techniques. We propose: 1) single-cell RNA sequencing (scRNAseq) of splenic T cells from mice that consumed milk±BTN1A1; 2) scRNAseq on T cells isolated from peripheral blood mononuclear cells (PBMCs) derived from healthy human donors and exposed to anti-CD3/28 ± recombinant BTN1A1; and 3) the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to define BTN1A1-dependent epigenetic changes in T cells. Aim 2 will test whether BTN1A1 mediates immune tolerance to environmental allergens in vivo. Exposing lactating mothers to Ova has been shown to generate tolerance to Ova in their suckling offspring, preventing Ova-induced reactive airway disease in those offspring when they are challenged with Ova after weaning. Using this experimental paradigm, we will test whether BTN1A1 is required for the protective effect of breast milk against Ova-induced lung disease. The proposed studies investigating the immune regulating functions of BTN1A1 will contribute important knowledge regarding how breast milk affects the neonatal immune system and lowers the risk of atopy and autoimmunity.